Currently, methods for analyzing a target gene of an analyte, that are widely conducted in a molecular diagnosis field, may be largely classified into two, a relative or absolute quantitative method of measuring expression degree of the target gene and the copy number thereof and a qualitative method of analyzing presence or absence of the target gene and the genotype thereof.
Among the methods for analyzing a target gene of an analyte, the real-time polymerase chain reaction (PCR) is a representative quantitative method, which is employed in measuring the expression degree of the target gene and the copy number thereof.
The real-time PCR method employs an apparatus where a PCR apparatus (thermal cycler) and a spectrophotometer are integrated with each other, and analyzes the amount of DNA or RNA fed or present at the early time by monitoring a procedure of producing amplification products of the target gene in real time during an amplification procedure, without a separate electrophoresis procedure for confirming PCR products after the PCR procedure. More specifically, the real-time polymerase chain reaction (PCR), where a fluorescent material is applied to the PCR method, may real-time detect and quantitatively analyze the degree of emission of the fluorescent material increasing in proportion to amplification of a target gene existing in the specimen during the reaction, thereby promptly and accurately analyzing the expression type of the target gene and the copy number thereof. As for the method of obtaining the fluorescent signal in the real-time polymerase chain reaction, there are largely a non-specific detection method and a specific detection method. As for the non-specific detection method, a fluorescent body (e.g., SYBR Green I, BEBO, BOXTO, EvaGreen, etc.) emitting a fluorescent material signal when being intercalated in the DNA double strands is used, and the entire fluorescent signal of double strands formed during the gene amplification is measured. As for the specific detection method, a fluorescent body is intercalated in a short oligonucleotide strand having a specific sequence that is complementarily bindable to a target gene to thereby form a probe (e.g., TaqMan probe, Molecular Beacon, Light-Up probe, Hybridization probe, or the like), and when the short oligonucleotide probe is amplified together with the target gene, a fluorescent signal generated from the short oligonucleotide probe in proportion to gene amplification is measured.
In the real-time polymerase chain reaction using the TaqMan probe in the specific detection method, a principle thereof is the same as that of a general PCR in view of using two primers, but an oligonucleotide probe chemically bound to a fluorescent material needs to be used. As for the TaqMan probe, FAM or the like as a reporter fluorescent material is attached to a 5′ end thereof and TAMRA or the like as a quencher material of offsetting a fluorescence of the reporter fluorescent material is attached to a 3′ end thereof. When the TaqMan probe is added to the PCR reactive solution, the TaqMan probe is annealed to the template DNA due to annealing with the template DNA during the PCR procedure, but the fluorescence is not detected by the offsetting action of the reporter and the quencher material. When the TaqMan probe hybridized with the template DNA is disintegrated due to activation of 5′. 3′ exonuclease of the Taq DNA polymerase at the time of extension by the Taq DNA polymerase during the PCR procedure, FAM or the like, which is a fluorescent material, is isolated to thereby cancel the offset by the quencher material, resulting in emitting a fluorescence. Hence, the amount of amplified products generated may be measured by analyzing the amount of fluorescence detected.
Meanwhile, in the real-time polymerase chain reaction (PCR) using SYBR Green I in the non-specific detection method, the entire fluorescence is monitored. This method employs a principle where a fluorescent material is intercalated in the double strand DNA chain. SYBR Green I, which is a fluorescent material, is added at the time of PPCR reaction, to thereby detect the amount of fluorescence emitting in proportion to amplification of PCR products, and thus, the generation amount of amplified products may be measured and the melting point of the amplified DNA may be measured.
In addition, as for qualitative method used in analyzing the presence or absence of a target gene and the genotype thereof among the methods of analyzing a target gene, there is recently a DNA microarray (DNA chip) method capable of simultaneously detecting several tens to several tens of hundreds of kinds of genes on one sheet of slide through the combination of molecular biological technology and electronic engineering technology.
More specifically, as a method of using a microarray-fixed chip where oligonucleotide, PAN, or cDNA, capable of detecting a target gene is used as a probe, there is a microarray chip. According to this method, the amplified products after the PCR reaction is hybridized with a target microarray chip, to thereby be bound to several kinds of probes (oligonucleotide, PAN, or cDNA) immobilized on a surface of the chip solid (glass, plastic, membrane, silicon, etc.), and then the presence or absence of the binding may be determined by a fluorescent material signal labeled on the amplified product, and thus, this method is useful in analyzing various types of genotypes. However, the test procedure is complicated and a somewhat large amount of time is required, and the results are not visually confirmable by the naked eyes to thereby require a separate fluorescent analysis apparatus.
Recently, in the case of diseases by infectious bodies in a molecular diagnosis field as above, as various concepts of diagnosis and therapy are applied, the method of analyzing a target gene of an analyte simultaneously and complexly requires quantitative information of the copy number for confirming the infectious degree through a real-time polymerase chain reaction and qualitative information of the genotype by utilizing a microarray chip for prescription of an appropriate therapeutical agent by confirming the kind of infectious body. According to this purpose, the real-time polymerase chain reaction and the microarray chip analysis method are independently used in combination with each other.
However, according to the combination of the analysis methods above, experiments for the analysis methods are sequentially carried out in different reactors, and analyzed, and thus, a large amount of labor and time are still required and inconvenience remains. In addition, it is difficult to automate the analysis methods in a single device. Even though an apparatus for automating the simple combination of the analysis methods is manufactured, a large space for the installing the apparatus is required and the apparatus is very expensive, and thus, it is not easy to routinely use the apparatus for molecular diagnosis in a hospital. Therefore, development of a technology for solving these problems is really needed.